Ganoderic acid A exerts antitumor activity against MDA-MB-231 human breast cancer cells by inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

Abstract

Breast cancer is a common malignant tumor among females, with triple-negative breast cancer being an important type accounting for 15-20% of all breast cancer cases. Triple-negative breast cancer is one of the most aggressive types of cancer without standard adjuvant chemotherapy. Ganoderic acid A (GA-A) is one of the major bioactive Ganoderma triterpenoids isolated from Ganoderma, which are recognized for their preventative and therapeutic effects. In the present study, the antineoplastic effect of GA-A on human breast cancer was investigated and the pro-apoptotic function of Janus kinase (JAK)2 and signal transducer and activator of transcription (STAT)3 on the function of GA-A was revealed. GA-A treatment inhibited the invasion of MDA-MB-231 cells. In addition, GA-A exhibited significant antitumor activity by enhancing the apoptotic index and reactive oxygen species production. In the present study, GA-A was identified to directly inhibit JAK2 phosphorylation and STAT3 downstream activation. In addition, GA-A suppressed STAT3 target gene expression, including B cell lymphoma-extra-large and Myeloid cell leukemia 1, resulting in elevated levels of proteins associated with mitochondrial apoptosis in addition to inhibitors of cyclin-dependent kinase. GA-A, in combination with AG490, a JAK2/STAT3 inhibitor, further decreased MDA-MB-231 cell viability. In conclusion, GA-A treatment inhibited breast cancer cell viability via JAK2/STAT3 downregulation and may regulate associated targets to serve an anti-MDA-MB-231 role, including mitochondrial apoptosis and regulating the expression of cell-cycle-associated factors.

Keywords: Ganoderic acid A; Janus kinase 2; apoptosis; signal transducer and activator of transcription 3; triple-negative breast cancer.

Figure 1.

Structure of Ganoderic acid A.

Figure 2.

GA-A inhibited the viability and invasive capacity of MDA-MB-231 cells. (A) GA-A inhibited MDA-MB-231 cell viability in a dose and time dependent manner. *P<0.05 vs. control group, ***P<0.001 vs. control. (B) GA-A inhibits MDA-MB-231 cell invasive capacity. GA-A, Ganoderic acid A.

Figure 3.

GA-A induced the apoptosis of MDA-MB231 cells. (A) GA-A induced MDA-MB-231 apoptosis as measured by flow cytometry and (B) quantified. **P<0.01 vs. control, ##P<0.01 vs. GA-A 0.1 mmol/l group, ŞŞP<0.01 vs. GA-A 0.2 mmol/l group. GA-A, Ganoderic acid A; PE, phycoerythrin; FITC, fluorescein isothiocyanate.

Figure 4.

GA-A upregulated reactive oxygen species levels in MDA-MB-231 cells. MDA-MB-231 cells were (A) untreated or treated with GA-A (B) 0.1 mmol/l, (C) 0.2 mmol/l and (D) 00.4 mmol/l for 24 h. GA-A, Ganoderic acid A.

Figure 5.

GA-A affected the expression of cell cycle association proteins cyclin D1, p21 and p27 in MDA-MB-231 cells. (A) Western blot analysis and (B) quantified relative expression. ***P<0.001, **P<0.01, *P<0.05 vs. GA-A-untreated control. GA-A, Ganoderic acid A; n.s., not significant.

Figure 6.

GA-A affected JAK2/STAT3 signaling pathway and mitochondrial apoptotic pathway associated proteins in MDA-MB-231 cells. (A) The bands of western blot analysis. β-actin was used as an internal control. (B) Statistical charts of the relative expression levels of t-JAK2 and p-JAK2. (C) Statistical charts of the relative expression levels of t-STAT3 and p-STAT3. (D) Statistical charts of the relative expression levels of Bcl-xL. (E) Statistical charts of the relative expression levels of Mcl-1. (F) Statistical charts of the relative expression levels of Bax. (G) Statistical charts of the relative expression levels of Bak. (H) Statistical charts of the relative expression levels of cytosolic Cytochrome c. ***P<0.001, **P<0.01, *P<0.05 vs. GA-A-untreated control. GA-A, Ganoderic acid A; STAT, signal transducer and activator of transcription; JAK, Janus kinase; Bcl-xL, B cell lymphoma-extra-large; Mcl-1, myeloid cell leukemia 1; Bax, Bcl2 associated X protein; Bak, Bcl2 antagonist/killer; n.s., not significant; p-, phosphorylated; t-, total.

Figure 7.

Effects of GA-A combined with AG490 on JAK2/STAT3 signaling. (A) The bands of western blot analysis. β-actin was used as an internal control. Relative expression levels of (B) p-JAK2 and (C) p-STAT3. ***P<0.001, **P<0.01, *P<0.05 vs. GA-A-untreated control. GA-A, Ganoderic acid A; STAT, signal transducer and activator of transcription; JAK, Janus kinase; n.s., not significant; p-, phosphorylated; t-, total.